H5N1型禽流感病毒HA基因克隆及序列分析

Cloning and Sequence Analysis of HA Gene of H5N1 Avian Influenza Virus

为从分子生物学角度进一步研究H5N1型禽流感主要抗原HA基因,探究H5N1HA基因的遗传变异情况及其蛋白结构,根据GenBank中登录的H5N1型禽流感HA基因的序列利用Primer5.0设计引物扩增HA基因,将其克隆到pMD18-T载体后进行鉴定,对鉴定正确的重组质粒pMD-H5N1-HA进行测序分析。应用生物信息学软件对禽流感病毒HA基因进行系统进化树分析、氨基酸序列同源性分析、保守区分析,磷酸化位点和序列重复区分析及HA蛋白二级、三级结构的预测。核苷酸序列测定结果表明:HA基因长1661bp,共编码326个氨基酸;生物信息学分析结果表明:HA基因在种间具有较高保守性,HA基因的氨基酸序列与其他地方性禽流感H5N1病毒株HA基因的序列同源性最高可达99.7%,且为高致病性毒株;HA蛋白的二级、三级结构预测结果显示:HA蛋白多为α螺旋和β转角结构,无规则卷曲,是亲水性蛋白。该结果为禽流感病毒的分子病毒学研究奠定了基础。

This research aimed to study HA gene of H5N1 avian influenza virus pathogen from the perspectiveof molecular biology and explore its genetic variation and protein structure. Primer 5.0 was used to design apair of primers according to the sequence of HA gene of H5N1 avian influenza virus published in Gen Bank. HAgene was amplified, cloned to p MD18-T vector and then identified. The recombinant plasmid p MD-AIV-HA,which was identified to be correct, was then analyzed by nucleotide sequence analysis. Bioinformatics softwarewas used to do phylogenetic analysis of HA gene, amino acid sequence homology analysis, conserved sequenceanalysis, phosphorylation sites analysis, protein repeated sequence analysis and the secondary and tertiarystructure prediction of HA protein. The sequencing results revealed that HA gene was 1661 bp and encoded326 amino acids. The analysis of bioinformatics indicated that HA gene was conserved in species, and aminoacid sequence homology between HA gene of H5N1 avian influenza virus and of other different AIV strainscould reach 99.7%. It was also a highly pathogenic strain. The secondary and tertiary structure predictionresults manifested that HA protein mostly consisted of α-helix, β-turn and random coil, and was a hydrophilic protein. The results would lay a foundation for the molecular virology research of AIV.